Swellable and structurally homogenous hydrogels and methods of use thereof

ABSTRACT

The invention encompasses hydrogels, monomer precursors of the hydrogels, methods for the preparation thereof, and methods of use therefor. The linking of monomers can take place using non-radical, bioorthogonal reactions such as copper-free click-chemistry.

RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.62/626,920, filed on Feb. 6, 2018. The entire teachings of the aboveapplication are incorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with government support under Grant No. 6936173awarded by ARO and under Grant No. 6934416 awarded by the NationalInstitutes of Health. The government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

Expansion microscopy (ExM), described for example in WO2015127183 andChen et al., Science, 347, 543 (2015), is a technique that allows forthree-dimensional (3D) nanoscale imaging of biological samples byphysically expanding the specimens¹⁻⁴. In ExM, hydrogels are synthesizedwithin the biological samples. During the gelation process, biomoleculesor tags are anchored to the hydrogel matrix. The hydrogel-specimencomposite then goes through a 3D expansion, physically separating thebiomolecules or tags.

In all of the ExM processes reported, the swellable hydrogel issynthesized by radical polymerization, a reaction known to introducestructural inhomogeneities at nanoscopic length scales⁵⁻⁷. Thestructural inhomogeneity is mainly caused by two factors: (a) localfluctuation of reagent concentrations during gelation and (b)topological defects such as loops and entanglements of polymer chains.

Therefore, there is a need in the art for a swellable hydrogel that isstructurally homogenous down to the nanoscopic length scale.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing (sequencelisting.txt;Size: 1,949 bytes; Date of Creation: Sep. 6, 2023) is hereinincorporated by reference in its entirety.

SUMMARY OF THE INVENTION

The invention encompasses hydrogels, monomer precursors of thehydrogels, composites comprising the hydrogel and a biological sample,methods for the preparation of the hydrogels and the composites, andmethods of using the hydrogels and the composites. As described in moredetail below, the hydrogels are designed to be structurally homogenousdown to the nanoscopic length scale. The linking of the monomersdescribed herein can take place using non-radical, bio-orthogonalreactions such as copper-free click-chemistry.

In some aspects, the hydrogel is the product of a non-radicalpolymerization reaction between a monomer of Formula A, wherein themonomer of Formula A is a monomer of Formula A1, Formula A2, Formula A3,Formula A4, Formula A5, or Formula A6.

In some aspects, the hydrogel is the product of a non-radicalpolymerization reaction between a monomer of Formula (A1):

and a monomer of Formula (B1):

wherein: each n is an integer greater than or equal to 1; each p is aninteger greater than or equal to 1; X and Y₁ are each crosslinkablemoieties; Z⁺ is a counter cation; and X and Y₁ covalently crosslink toend-link the monomers. In preferred aspects, the non-radicalpolymerization is bio-orthogonal. In certain aspects, X is a moietycomprising a terminal azide group and Y₁ is a moiety comprising aterminal alkyne and X and Y₁ crosslink by copper-free azide-alkynecycloaddition.

In additional aspects, the hydrogel is the product of a non-radicalpolymerization reaction between a monomer of Formula (A2):

and a monomer of Formula (B2)

wherein: each n is an integer greater than or equal to 1; each q is aninteger greater than or equal to 1; Y₂ is a moiety comprising a terminaldibenzocyclooctyl (DBCO) or a terminal bicyclononyne; X₁ is a moietycomprising a terminal azide group; Z⁺ is a counter cation; and X₁ and Y₂crosslink by copper-free azide-alkyne cycloaddition. In preferredaspects, the non-radical polymerization is bio-orthogonal. In certainaspects, Z⁺ is Na⁺ or K⁺.

In certain additional aspects, Z⁺ is Na⁺ or K⁺. A non-limiting exampleof a monomer of Formula (A1) has the Formula (A3):

Thus, the invention also encompasses a hydrogel that is the product of anon-radical polymerization reaction between a monomer of Formula (A3)and a monomer of Formula (B1).

A non-limiting example of a monomer of Formula (A2) has the Formula(A4):

The invention also includes a hydrogel that is the product of anon-radical polymerization reaction between a monomer of Formula (A4)and a monomer of Formula (B2).

The invention additionally encompasses a hydrogel that is the product ofa non-radical polymerization reaction between a monomer of Formula (A5):

and the monomer of Formula (B1) as described above, wherein E is amoiety comprising a charged functional group and Z is a counter ion (forexample, a counter cation or counter anion depending on the charge ofE); and X and n are as defined above for Formula (A1). In certainaspects, E is a charged functional group; for example, E is selectedfrom a carboxylic acid group, an ammonium group, and a sulfate group.

The invention additionally encompasses a hydrogel is the product of anon-radical polymerization reaction between a monomer of Formula (A6):

and the monomer of Formula (B2) as described above, wherein E and Z areas defined above for Formula (A5); and X and n are as defined above forFormula (A1). In certain aspects, E is selected from a carboxylic acidgroup, an ammonium group, and a sulfate group.

In certain additional aspects, the invention is directed to a compositecomprising a biological sample and a hydrogel that is the product of anon-radical polymerization reaction between the monomer of Formula (A1),(A2), (A3), (A4), (A5) or (A6) (collectively referred to herein asFormula (A)) and the monomer of Formula (B1). The invention alsoencompasses a method of preparing the composite comprising permeatingthe biological sample with the monomer of Formula (A1), (A2), (A3),(A4), (A5) or (A6) and the monomer of Formula (B1) under conditionssuitable to form a hydrogel by non-radical polymerization.

In certain additional aspects, the invention is directed to a compositecomprising a biological sample and a hydrogel that is the product of anon-radical polymerization reaction between the monomer of Formula (A2),(A4), or (A6) and the monomer of Formula (B2). The invention alsoencompasses a method of preparing the composite comprising permeatingthe biological sample with the monomer of Formula (A2), (A4), or (A6),and the monomer of Formula (B2) under conditions suitable to form ahydrogel by non-radical polymerization. In yet additional aspects, theinvention is directed to a method of microscopy comprising:

-   -   a. permeating the biological sample with a monomer of Formula        (A1), (A2), (A3), (A4), (A5) or (A6) and a monomer of Formula        (B1) under conditions suitable to form a hydrogel by non-radical        polymerization;    -   b. isotropically expanding the composite by contacting it with        an aqueous solution; and    -   c. viewing the expanded composite using microscopy.

In further aspects, the invention is directed to a method of microscopycomprising:

-   -   a. permeating the biological sample with a monomer of Formula        (A2), (A4), or (A6), and a monomer of Formula (B2) under        conditions suitable to form a hydrogel by non-radical        polymerization;    -   b. isotropically expanding the composite by contacting it with        an aqueous solution; and    -   c. viewing the expanded composite using microscopy.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided to the Office upon request and paymentof the necessary fee.

The foregoing and other objects, features and advantages of theinvention will be apparent from the following more particulardescription of preferred embodiments of the invention, as illustrated inthe accompanying drawings in which like reference characters refer tothe same parts throughout the different views. The drawings are notnecessarily to scale, emphasis instead being placed upon illustratingthe principles of the invention.

FIGS. 1A and 1B: Design of swellable, structurally homogenous hydrogelsbased on tetrahedral monomers. (a) Tetrahedral monomers A and B havefunctional end groups that specifically and complementarily bind to eachother. (b) One implementation of Monomer A with repeated sodium acrylateunits.

FIG. 2 : Synthesis of one version of Monomer A (shown in FIG. 1B) basedon the 4-arm sodium polyacrylate structure. The synthesis starts witharm elongation via atom transfer radical polymerization (syntheticdetails shown as “*”) of tert-butyl acrylate. The bromide end groups arefurther modified to azide end groups for copper click-chemistry used tolink the monomers. The tert-butyl polyacrylate arms are then deprotectedto yield Monomer A.

FIGS. 3A and 3B: Synthesis of one version of Monomer B based on 4-armPEGs. (a) Using 4-arm PEG-amines as the starting material, different endgroups such as (i) dibenzocyclooctyl (DBCO), (ii) bicyclononyne (BCN) or(iii) dibenzocyclooctyl-disulfide (DBCO-SS) groups can be added to thearm ends via amine/NHS ester reaction. (b) An intermediate in theMonomer A synthesis (in FIG. 2 ) can be further modified into a 4-armsodium polyacrylate species with DBCO end groups and can be used asMonomer B.

FIG. 4 : Synthesis and expansion of hydrogel (termed as “tetragel”)formed by reacting Monomer A and Monomer B. Fluorescence images oftetragels after expansion using three different Monomers B with the endgroups specified in FIG. 3 a-i (DBCO), 3 a-ii (BCN) and 3 a-iii(DBCO-SS) are shown. Fluorescent image of the tetragel before expansion(the pre-expansion size is the same for all three versions of Monomer B)is shown on the left for comparison. About 1 mol % fluorescein amine wasanchored to the hydrogel matrix with NHS-azide to fluorescentlyvisualize the gels. Scale bar, 5 mm.

FIG. 5A: In situ synthesis and expansion of brain tissue with tetragel.Fluorescence images of the same Thy1-YFP mouse brain slice before (left)and after (right) expansion with tetragel. The post-expansion sample wasimmunostained by anti-GFP primary antibodies and dye-conjugatedsecondary antibodies. Scale bar: 2 mm (left) and 6 mm (right).

FIG. 5B: Post-expansion Thy1-YFP mouse brain slices. Scale bar, 10 μm(right, 30 μm)

FIG. 6A-6G: In situ synthesis and expansion of cultured cells withtetragel. (a, c) Fluorescence image of expanded HEK 293 cellsimmunostained with anti-α-tubulin primary antibodies and dye-conjugatedsecondary antibodies. Scale bar, 20 μm. (b, d) Fluorescence image of thesame cells before expansion. Scale bar, 20 μm. (e) Line profile of theimage specified by the solid line in (c). (f) Line profile of the imagespecified by the solid line in (d). (g) RMS error curve for the HEK293cell expansion (blue line, mean; shaded area, standard deviation; n=8cells). Inset, non-rigidly registered and overlaid pre-expansion (green)and post-expansion (magenta) images used for the RMS error estimation.

FIG. 7A-7C: In situ synthesis and expansion of nuclear pore complex withtetragel. (a) Pre-expansion live fluorescent image of nuclear porecomplex with GFP fusion. Scale bar, 10 μm. (b), (c) Post-expansionfluorescent image of (b) GFP and (c) Cy3-conjugated secondary antibody(Nup133 primary antibody). Scale bars, 20 μm.

FIGS. 8A and 8B: Retention of far-red dye in tetragel. (a) Fluorescenceimages of mouse brain slices stained with far-red dyes before and afterexpansion with tetragel. The brain slices were immunostained with Tom 20primary antibodies and far-red dye conjugated secondary antibodies. (b)Retention of far-red dyes (Alexa Fluor 647 (AF647) and Cyanine 5 (Cy5))with Stock X gel (used for conventional ExM) and tetragel.

FIG. 9A: Pre-expansion (left column) and post-expansion (right column)Thy1-YFP mouse brain slices immunostained with TOMM20 primary antibodiesand AF647-conjugated secondary antibodies, using polyacrylamide/sodiumpolyacrylate gels (PAAs, top row) and TGs (bottom row). Scale bars, 300μm (top right, 1.18 mm; bottom right, 815 μm).

FIG. 9B: Fluorescence retention of far-red and infra-red dyes (AF647,Cy5 and AF680) using PAAs and TGs.

FIG. 10 : Design and synthesis of structurally homogenous hydrogelmatrix for expansion of nanoscale biological structures. a,Cell/tissue-hydrogel composites formed by in situ free-radicalpolymerization have structural inhomogeneities at 10-100 nm length scaledue to (1) the local fluctuation of monomer and cross-linking density,and (2) the dangling ends and (3) loops in the polymer chains. b, Designof monomers 1 and 2 (monomers A and B; and referred to in the Examplesas “monomer 1” and “monomer 2”) with tetrahedral symmetry and reactiveterminal groups. Modification of monomer 2 terminal groups (2, 2′ and2″) allows fine tuning of the reactivity between the monomers andadditional functionality of, for example, cleavability. c, Formation andexpansion of tetra-gels (TGs) via click chemistry-based terminal-linkingof monomers 1 and 2 (or 2′, 2″). Inset, projected planar view of thepolymerized network.

FIG. 11A-11D: Tetragel (TG)-based iterative expansion enablesnanoscopically isotropic expansion at 10-100 nm length scale. a, ShortDNA-oligos (22 base pairs) were directly anchored to the envelopeproteins of an HSV-1 virion via hydrazone formation. The DNA-oligos wereused for signal transferring, amplification, and fluorescence readout inthe subsequent iterative expansion process. b, HSV-1 virions witholigo-labeled envelope proteins, expanded by TG-based (left) andPAA-based (right) two-round iterative expansion. Scale bar, 1 μm (10.3μm and 15.3 μm for TG and PAA, respectively). c, Left, averagedsingle-particle images of HSV-1 virions by TG-based (top) and PAA-based(bottom) expansion. Scale bars, 100 nm. Right, normalized fluorescenceintensity along the dotted lines. d, 3D rendered images of an HSV-1virion particle with the oligo-labeled envelope proteins, expanded byTG-based three-round iterative expansion. The raw data (white, left)overlaid with the fitted centroids (red, left) and the extractedcentroids (colored, right) are shown. Scale bars, 100 nm (3.83 μm).Inset, maximum intensity projection (MIP) of the same virion particleover a ˜65 nm range close to the particle center (as shown between thetwo lines in the rendered image). Scale bars, 100 nm (3.83 μm).

FIG. 12 : HSV-1 virions with oligo-labeled envelope proteins (white) andDAPI-labeled DNAs (blue). The virions were expanded by TG-based 10-folditerative expansion. Scale bar, 1 μm (10.7 μm).

DETAILED DESCRIPTION OF THE INVENTION

A description of preferred embodiments of the invention follows.

As used herein, the words “a” and “an” are meant to include one or moreunless otherwise specified. For example, the term “a cell” encompassesboth a single cell and a combination of two or more cells.

As will be apparent to those of skill in the art, each of the individualembodiments described and illustrated herein has discrete components andfeatures which can be readily separated from or combined with thefeatures of any of the other several embodiments without departing fromthe scope or spirit of the present teachings. Any recited method can becarried out in the order of events recited or in any other order whichis logically possible.

As used herein, the terms “specimen” or “sample” are usedinterchangeably herein and include, but are not limited to tissues,including but not limited to, liver, spleen, kidney, lung, intestine,thymus, colon, tonsil, testis, skin, brain, heart, muscle and pancreastissue. Other exemplary biological samples include, but are not limitedto, biopsies, bone marrow samples, organ samples, skin fragments andorganisms. Materials obtained from clinical or forensic settings arealso within the intended meaning of the term biological sample. In oneembodiment, the sample is derived from a human, animal, or plant. In oneembodiment, the biological sample is a tissue sample, preferably anorgan tissue sample. In one embodiment, samples are human. The samplecan be obtained, for example, from autopsy, biopsy or from surgery. Itcan be a solid tissue such as, for example, parenchyme, connective orfatty tissue, heart or skeletal muscle, smooth muscle, skin, brain,nerve, kidney, liver, spleen, breast, carcinoma (e.g., bowel,nasopharynx, breast, lung, stomach etc.), cartilage, lymphoma,meningioma, placenta, prostate, thymus, tonsil, umbilical cord oruterus. The tissue can be a tumor (benign or malignant), cancerous orprecancerous tissue. The sample can be obtained from an animal or humansubject affected by disease or other pathology or suspected of same(normal or diseased), or considered normal or healthy. The biologicalsample can, for example, be a cell sample. In certain aspects, thebiological sample is a virus or virion. The term “biological sample” cana biological sample that comprises, or is believed to comprise, nucleicacid sequences including, but not limited to cDNA, mRNA and genomic DNA.

Tissue specimens suitable for use with the methods and systems describedherein generally include any type of tissue specimens collected fromliving or dead subjects, such as, e.g., biopsy specimens and autopsyspecimens. Tissue specimens may be collected and processed using themethods and systems described herein and subjected to microscopicanalysis immediately following processing, or may be preserved andsubjected to microscopic analysis at a future time, e.g., after storagefor an extended period of time. In some embodiments, the methodsdescribed herein may be used to preserve tissue specimens in a stable,accessible and fully intact form for future analysis. For example,tissue specimens, such as, e.g., human brain tissue specimens, may beprocessed as described above and cleared to remove a plurality ofcellular components, such as, e.g., lipids, and then stored for futureanalysis.

Tissues that have been preserved, or fixed, contain a variety ofchemical modifications that can reduce the detectability of proteins inbiomedical procedures. In some embodiments, the methods and systemsdescribed herein may be used to analyze a previously-preserved or storedtissue specimen. Previously preserved tissue specimens include, forexample, clinical samples used in pathology including formalin-fixedparaffin-embedded (FFPE), hematoxylin and eosin (H&E)-stained, and/orfresh frozen tissue specimens. If the previously preserved sample has acoverslip, the coverslip should be removed. The sample is treated toremove the mounting medium. Such methods for removing the mountingmedium are well known in the art. For example, treating the sample withxylene to remove paraffin or other hydrophobic mounting medium.Alternatively, if the sample is mounted in a water-based mountingmedium, the sample is treated with water. The sample is then rehydratedand subjected to antigen-retrieval. The term “antigen retrieval” refersto any technique in which the masking of an epitope is reversed andepitope-antibody binding is restored such as, but not limited to, enzymeinduced epitope retrieval, heat induced epitope retrieval (HIER), orproteolytic induced epitope retrieval (PIER). For example, the antigenretrieval treatment can be performed in a 10 mM sodium citrate buffer aswell as the commercially available Target Retrieval Solution(DakoCytomation) or such.

The term “bio-orthogonal” in reference to a chemical reaction refers toa chemical reaction that does not interfere with any other chemicalmoieties in the natural or native surroundings.

The term “sequencing,” as used herein, refers to a method by which theidentity of at least 10 consecutive nucleotides (e.g., the identity ofat least 20, at least 50, at least 100 or at least 200 or moreconsecutive nucleotides) of a polynucleotide is obtained.

Monomers of Formulae (A1), (A2), (A3), (A4), (A5) and (A6) cancollectively be referred to as monomers of Formula (A). Monomers ofFormula (B1) and (B2) can collectively be referred to as monomers ofFormula (B).

The term “hydrogel AB1” is used to refer to a hydrogel that is theproduct of a reaction between a monomer of Formula (A) and a monomer ofFormula (B1), and the term “hydrogel AB2” is used to refer to a hydrogelthat is the product of a reaction between a monomer of Formula (A) and amonomer of Formula (B2). Other hydrogel that are the product of areaction between a monomer of Formula (A) and a monomer of Formula (B1)or (B2) can be similarly referred to.

The invention encompasses hydrogels, monomer precursors of thehydrogels, methods for the preparation of the hydrogels, and methods ofusing the hydrogel, for example, in expansion microscopy and/or in situsequencing, wherein the hydrogel is the product of a non-radicalpolymerization reaction between a monomer of Formula (A) and a monomerof Formula (B1), or the product of a non-radical polymerization reactionbetween a monomer of Formula (A2), (A4), or (A6) and a monomer ofFormula (B2).

In certain aspects, the invention is directed to a hydrogel that is theproduct of a non-radical polymerization reaction between a monomer ofFormula (A1), (A2), (A3), (A4), (A5), or (A6) and a monomer of Formula(B1). In certain additional aspects, the X of Formula (A1), (A3), and/or(A5) or X₁ of Formula (A2), (A4), or (A6) is azide (—N₃) and the Y₁ ofFormula (B1) is a cyclic alkyne.

In additional aspects, the invention is directed to a hydrogel that isthe product of a non-radical polymerization reaction between a monomerof Formula (A2), (A4), or (A6), and a monomer of Formula (B2).

The invention also encompasses a monomer of Formula (A1), (A3), and(A5), in certain embodiments, X is azide. The invention additionallyencompasses a monomer of Formula (A2), (A4), and (A6), wherein X₁ isazide. The invention additionally encompasses a monomer of Formula (B1);in certain aspects, Y₁ is a cyclic alkyne such as dibenzocyclooctyl(DBCO) or a bicyclononyne. In further aspects, the invention encompassesa monomer of Formula (B2); in certain aspects, X is azide.

In certain aspects, E of Formula (A5) or Formula (A6) is a negativelycharged functional group. Exemplary negatively-charged groups include,without limitation, carboxylic (e.g., acetic) group, sulfo group,sulfino group, phosphate group and phosphono group. In yet additionalaspects, E of Formula (A5) or Formula (A6) is a positively chargedfunctional group. Examples of positively-charged functional groupsinclude, without limitation, amino (amine) groups that can be protonatedto form an ammonium group. In certain aspects, a charged functionalgroup is one that exhibits a charge at, or near, neutral pH (pH of about5 to about 9 or about 6 to about 8) in an aqueous medium.

Z is a counter ion. For example, in Formulae (A5) and (A6), if Ecomprises a negatively charged functional group, then Z is a countercation, and if E comprises a positively charged functional group, then Zis a counter anion.

Z⁺ is a counter cation such as an alkali metal atom, an alkali earthmetal atom, or substituted or unsubstituted ammonium. Non-limitingexamples of counter cations are potassium, sodium, mercury, lithium,magnesium, calcium, butylammonium, trimethylammonium, and tetramethylammonium. In certain aspects, the counter cation is sodium or potassium(Na+ or K+). In yet additional aspects, the counter cation is (Na+). Incertain aspects, the monomer of Formula (A1) has the Formula (A3):

The hydrogels described herein are swellable and can be used inexpansion microscopy (ExM). In ExM, chemically fixed and permeabilizedtissue (or other biological sample) is infused with swellable material,undergoes polymerization, and the tissue-polymer composite is treated tohomogenize its mechanical characteristics. Next, dialysis in water oraqueous solution results in isotropic expansion, thereby achievingsuper-resolution with diffraction-limited microscopes, and enablingrapid image acquisition and large field of view (Chen et al., Science,347, 543 (2015)). Expansion allows individual nucleic acids, normallydensely packed, to be resolved spatially in a high-throughput manner.Furthermore, the expanded environment is 99% water, facilitating enzymeaccess and creating “quasi-in vitro” environment while retaining spatialinformation. In some examples, fixation of the biological sample can beeffected by embedding the sample in a swellable material that has beenperfused throughout the sample as described by Chen et al. (Chen et al.,Science, 347, 543 (2015) and U.S. Patent Publication Nos. US20160116384-A1; US 20160305856-A1; US 20160304952-A1; and U.S. PatentPublication Nos. US 20170067096 A1 and US 20170089811 A1, eachcorresponding to U.S. patent application Ser. Nos. 15/229,539 and15/229,545, respectively, the contents of each of which are incorporatedherein by reference in their entirety. Briefly, a sample, such astissue, can be permeabilized. A permeabilized sample, or tissue, can beinfused with monomers or precursors of a swellable material and thencausing the monomers or precursors to undergo polymerization within thesample to form the swellable material. During or after polymerization,the swellable material can be anchored to the sample. Thesample-hydrogel complex (or composite) is optionally treated tohomogenize the mechanical characteristics of the sample. Thesample-swellable material complex can then be treated by dialysis in asolvent or liquid, such as in water, resulting in isotropic physicalexpansion of the sample. In this manner, the fixed biological sample isphysically “enlarged”, or “expanded”, as compared to the biologicalsample before swelling.

The swellable hydrogels currently being used in expansion microscopy(ExM) are synthesized by radical polymerization which is known tointroduce structural inhomogeneities at nanoscopic length scale. Thestructural inhomogeneity is mainly caused by two factors: (a) localfluctuation of reagent concentrations during the gelation and (b)topological defects such as loops and entanglements of polymer chains.To eliminate these intrinsic structural inhomogeneities, the hydrogelsdescribed herein have been designed which are structurally homogenousdown to the nanoscopic length scale. For example, two types ofpre-synthesized tetrahedral monomers are linked in a diamondlattice-like structure. The linking of the monomers takes place usingnon-radical, bio-orthogonal reactions including, but not limited to,copper-free click-chemistry. In this new hydrogel design, thehomogeneity in monomer shape and size mitigates the effect of reagentconcentration variations described in (a) above, resulting in a moreuniform distribution of monomers and cross-links throughout the gel.Furthermore, the specific and complementary linking chemistry betweenthe monomers reduces topological defects caused by (b) and thusfacilitates formation of a homogeneous and isotropic polymer network.

It has been shown that a non-swellable hydrogel with similar diamondlattice-like structure to the hydrogel described herein is structurallyhomogeneous and has nearly zero defects'. Specifically, Sakai andcolleagues demonstrated that end-linked tetrahedral monomers based onpolyethylene glycol (PEG) can form highly homogeneous hydrogel networksfree of defects'. These hydrogels, however, exhibit minimal expansion(˜1.3× volume) and thus cannot be directly used in ExM. Oshima andcolleagues investigated expandable hydrogels based on chargedtetrahedral polyacrylate monomers, finding that this network isstructurally superior to polyacrylate gels formed via radicalpolymerization in terms of sol fractions, dangling chains and trappedentaglements⁹. However, this strategy for gel formation required (1)copper catalysis and (2) treatment with trifluoroacetic acid, which areunlikely to be compatible with biological specimens.

In contrast to the gels of Sakai and Oshima, the hydrogels of thepresent invention are capable of isotropically expanding and areprepared using non-radical, bio-orthogonal reactions to end-linkmonomers. As described above, the lengths between cross-linkers (i.e.,the “mesh size”) need to be uniform in order to eliminate structuralinhomogeneities in hydrogels. In addition, topological defects, such asentanglements, loops and dangling chains, need to be significantlyreduced. The use of pre-synthesized monomers of defined arm lengthswhich are linked in a periodic fashion results in the formation of ahydrogel with structural homogeneity. For instance, two kinds ofhomogeneous tetrahedral monomers (Monomer A and Monomer B shown in FIG.1A) can be linked to form a diamond lattice-like polymer network (FIG.1A). In this design, monomers of Formula (A) and monomers of Formula(B1) or (B2) have specific and complementary linkers that bind two armsin between the two monomers. The linking needs to be covalent so thatwhen the hydrogel physically expands in water, the monomers will staylinked and the diamond lattice-like structure will be maintained.

In swellable hydrogels (in particular, those swelling in water), thepolymer chains commonly have charged functional groups so that thecharges can repel each other and pull the polymer chains apart. Forinstance, in the polyacrylamide/sodium polyacrylate hydrogels that havebeen described for use in ExM, the polymer chains have carboxylategroups that are negatively charged in water (“polyelectrolyte”). Therepulsion between the negative charges help to keep the hydrogelexpanded in water. In an example of the present design of tetrahedralmonomers, the monomers (specifically, the monomers of Formula (A) and/ormonomers of Formula (B2)) include repeats of sodium acrylate groups ineach arm to make the monomer charged in water. For example, a monomer ofFormula (A) and/or a monomer of Formula (B2) can include repeats ofsodium acrylate units in each arm of the monomer (FIG. 1B). Thehydrogels can also be prepared using a monomer of Formula (A) thatincludes repeats of ionized or charged functional groups other thancarboxylate. Such monomers are encompassed by Formulae (A5) and (A6).For example, the repeating functional group can be ammonium or sulfate.

The mesh size of the hydrogel can be systematically controlled bysynthetically changing the arm lengths of the monomers (“n” in theFormula (A), “p” in Formulae (B1) and “q” in Formula (B2)). In certainaspects, n of the Formulae (A) is 1 to 100. In yet additional aspects, nis 4, or 10, or 20, or 40. In yet additional aspects, the monomer ofFormula (A) has a molecular weight from about 0.5 kDa to about 50 kDa,or about 1 kDa to about 40 kDa, or about 1 kDa to about 25 kDa. By wayof example, the monomer of Formula (A3), wherein n is 4, 10, 20 or 40,has an approximate molecular weight of 2 kDa, 5 kDa, 10 kDa, and 20 kDa,respectively.

In certain additional aspects, p of Formula (B1) is 1 to 100. In yetadditional aspects, p of Formula (B1) is 36, or 72, or 144. In yetadditional aspects, the monomer of Formula (B1) has a molecular weightfrom about 10 kDa to about 300 kDa, or about 15 kDa to about 200 kDa, orabout 15 kDa to about 150 kDa. By way of example, the monomer of Formula(B1), wherein p is 36, 72 and 144, the monomer has an approximatemolecular weight of about 5 kDa, 10 kDa, and 20 kDa, respectively.

In certain additional aspects, q of Formula (B2) is 1 to 100. In yetadditional aspects, q is 36, 72, or 144.

As described above, the two monomers (a monomer of Formula (A) and amonomer of Formulae (B1) or (B2)) are end-linked to form a lattice-likepolymer network. Each of X and Y₁, X₁ and Y₁, X and Y₂, and X₁ and Y₂are complementary, reactive end-groups that are capable of forming acovalent bond. For example, in certain aspects, X is a moiety comprisinga terminal azide group and Y₁ is a moiety comprising a terminal alkyne,for example, a cyclic alkyne, and X and Y₁ crosslink by copper-freeazide-alkyne cycloaddition. In certain additional aspects, X or X₁ isazide (—N₃). Y₁ can, for example, be a cyclic alkyne moiety such as amoiety comprising a terminal dibenzocyclooctyl (DBCO) or a terminalbicyclononyne. Exemplary cyclic alkyne moieties include, but are notlimited to, DBCO, DBCO-SS (dibenzocyclooctyl-disulfide), DBCO-amine,DBCO-N-hydroxsuccinimidyl ester,(1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethyl N-succinimidyl carbonateand DBCO-maleimide. In yet additional aspects, Y₁ is selected from thegroup consisting of:

The linking chemistry between the monomers is not limited to thecatalyst-free click-chemistry described above. Other linking chemistriesinclude other bio-orthogonal reactions including, but not limited to,amine-NETS reaction, maleimide-thiol reaction, andtrans-cyclooctene/s-tetrazine reaction. Therefore, in some embodiments,wherein X and Y₁ crosslink by amine-NETS ester reaction. For example, Xcan be a moiety comprising a terminal amine and Y₁ can be a terminalN-hydroxysuccinimide ester group. Alternatively, X can be a moietycomprising a terminal N-hydroxysuccinimide ester group and Y₁ can be aterminal amine. In another example, X and Y₁ crosslink bymaleimide-thiol reaction. For example, X can be a moiety comprising aterminal sulfhydryl group and Y₁ can be a moiety comprising a terminalmaleimide group. Alternatively, X can be a moiety comprising a terminalmaleimide group and Y₁ can be a moiety comprising a terminal sulfhydrylgroup. In yet an additional example, X and Y₁ crosslink bytrans-cyclooctene (TCO)-tetrazine reaction.

In one example, the Monomer A (a monomer of Formula (A)) as shown inFIG. 1B can be synthesized via atom transfer radical polymerization(ATRP) and subsequent synthetic modifications (FIG. 2 )⁹. First,tert-butyl acrylates are added to a 4-arm tetrahedral core to thedesired arm length via ATRP (“*” in FIG. 2 ). In contrast to theconventional radical polymerization, ATRP offers site-specific andcontrolled addition of acrylate units to the growing polymer chains. Theend groups of the monomer arms are then modified from bromide to azidefor copper-free click chemistry. Finally, tert-butyl groups on themonomer arms are deprotected by a strong acid to yield Monomer A with4-arm sodium polyacrylate structure.

Monomer B (a monomer of Formula (B1)) can be synthesized byend-functionalization of commercially available 4-arm PEG-amines (FIG.3A). The end groups of the 4-arm PEG monomers can be modified to, forexample, (i) dibenzocyclooctyl (DBCO), (ii) bicyclononyne (BCN) or (iii)dibenzocyclooctyl-disulfide (DBCO-SS) groups, all of which are reactiveto the azide groups of Monomer A via copper-free click chemistry. BCNreacts more slowly with azide than DBCO or DBCO-SS and thus is moresuitable for biological samples that require longer diffusion timethrough the specimens before the gelation begins, such as intact tissuesamples. DBCO-SS can be cleaved by, for example, dithiothreitol (DTT).Therefore, the synthesized gel can be broken down at monomer level afterexpansion, making it compatible with processes that require breaking ofthe first hydrogel, such as the iterative ExM (iExM) process⁴ described,for example in U.S. Pat. App. Pub. No. 20160305856A1, the contents ofwhich are expressly incorporated by reference herein. Other monomers ofFormula (B1) can be prepared using a similar process.

A modified version of 4-arm sodium polyacrylate (a monomer of Formula(B2)) can also be used as Monomer B when its end groups are modified tofunctional groups that are reactive to azide, such as DBCO (FIG. 3B).Other monomers of Formula (B2) can be prepared using a similar process.Using a monomer of Formula (B2), the expansion factor of the hydrogelcan potentially be even larger than a hydrogel using a monomer ofFormula (B1) due to the increased negative charges on both monomers inwater.

The synthesis of the hydrogel takes advantage of the specific andcomplementary reaction between the monomers. For example, using MonomerA in FIG. 2B and Monomer B in FIG. 3A, a hydrogel (also termed a“tetragel”) can be formed by simply mixing the two monomers in water.The click-reaction between the two monomers proceeds without coppercatalyst. As an example, 10 uL of 4-arm sodium polyacrylate with azideend groups (200 mg/mL water, molecular weight ˜10 kDa, shown in FIG.2B), 10 uL of 4-arm PEG with BCN end groups (200 mg/mL DMSO, molecularweight ˜10 kDa, shown in FIG. 3 a-i ), and 40 uL of water are mixed andthen gelled at 37° C. for 2 hours. The synthesized hydrogel expands by afactor of about three-times in water (FIG. 4 ). As described above, thelinking chemistry between the monomers is not limited to thecatalyst-free click-chemistry described above. Other linking chemistriesinclude, but are not limited to, amine-NETS reaction, maleimide-thiolreaction, and trans-cyclooctene/s-tetrazine reaction.

The invention encompasses a composite comprising a biological sample anda hydrogel described herein (e.g., the hydrogel AB1 or the hydrogelAB2). In certain aspects, a biological sample can be embedded in ahydrogel as described herein. “Embedding” a sample in a swellablematerial or a swellable hydrogel comprises permeating (such as,perfusing, infusing, soaking, adding or other intermixing) the samplewith the swellable material, preferably by adding precursors thereof.The sample may be permeated (such as, perfusing, infusing, soaking,adding or other intermixing) with the precursors of the swellablematerial, wherein the sample is saturated with precursors of theswellable material, which flow between and around biomoleculesthroughout the specimen. Polymerizing the monomers or precursors isinitiated to form the swellable material or polymer in situ, wherein thepolymer network is formed within and throughout the specimen. In thismanner the biological sample is embedded in the swellable material.

The invention encompasses a method of preparing a composite comprising abiological sample and a hydrogel described herein comprising permeatingthe sample, for example, a biological sample, with a monomer of Formula(A), for example a monomer of formula (A1), (A2), (A3), (A4), (A5), or(A6), and a monomer of Formula (B1), or a monomer of Formula (A2), (A4)or (A6), and a monomer of Formula (B2) under conditions suitable to forma hydrogel by non-radical polymerization; and isotropically expandingthe composite by contacting it with an aqueous solution. The monomer ofFormula (A) and Formula (B1) or Formula (B2) can be added to the samplein separate solutions or as part of a single solution (for example,similar to the gelling solution described in the Examples). Permeatingthe sample entails, for example, perfusing, infusing, soaking, adding,or otherwise intermixing) with the monomers or with the precursors ofthe hydrogel. In order to prepare the composite, the precursors (forexample, a monomer of Formula (A) and monomer of Formula (B1)) can bereacted to form a hydrogel in situ. The monomers, can for example, be insolution, such as an aqueous solution. The solution can be a highconcentration solution, such as about 50% or more saturation (definedherein as the percentage of solids present in the aqueous solvent in thesame ratio as would result in precipitation under the conditions ofpermeation). In certain aspects, the solution is at high concentration,such as about 75% or more saturation, or 90% or more saturation.

In certain embodiments, the biological sample is permeated with themonomers, solutions comprising the monomers or hydrogel precursors, or asolution comprising the monomers (a monomer of Formula (A) and a monomerof Formula (B1), or a monomer of Formula (A) and a monomer of Formula(B2)) which are reacted to form a swellable polymer.

The hydrogels described herein are swellable. As used herein, the term“swellable” in reference to a hydrogel generally refers to a hydrogelthat expands when contacted with a liquid, such as water or othersolvent. In one embodiment, the swellable hydrogel of the presentinvention uniformly expands in three (3) dimensions. Additionally oralternatively, the hydrogel can be transparent such that, uponexpansion, light can pass through the sample. In one embodiment, theswellable hydrogel is formed in situ from precursors thereof.

In certain embodiments, the biological sample, or a labeled sample (asdescribed in more detail below), can, optionally, be treated with adetergent prior to being contacted with the precursors or monomers. Theuse of a detergent can improve the wettability of the sample or disruptthe sample to allow the precursor or monomers to permeate throughoutsample.

An expandable biological sample can be prepared by contacting the samplewith a bi-functional linker comprising a binding moiety and an anchor,wherein the binding moiety binds to biomolecules in the sample;permeating the sample with a composition comprising precursors of aswellable hydrogel; and initiating polymerization to form a swellablehydrogel, wherein the biomolecules are anchored to the swellablehydrogel to form a sample-swellable hydrogel complex. The precursors ofa swellable hydrogel comprise the monomers as described herein (i.e., amonomer of Formula (A) and a monomer of Formula (B1), or a monomer ofFormula (A) and a monomer of Formula (B2)), which are reacted to form aswellable hydrogel.

In one embodiment, the method for preparing an expandable biologicalspecimen comprises the steps of treating the specimen with abifunctional crosslinker; permeating the specimen with precursors of aswellable polymer; polymerizing the precursors to form a swellablepolymer within the specimen; and incubating the specimen with anon-specific protease in a buffer comprising a metal ion chelator, anonionic surfactant, and a monovalent salt. In one embodiment, themethod can further comprise the step contacting the sample withmacromolecules that will bind to biomolecules within the sample. Theprecursors of a swellable hydrogel comprise the monomers as describedherein (i.e., a monomer of Formula (A) and a monomer of Formula (B1), ora monomer of Formula (A) and a monomer of Formula (B2)), which arereacted to form a swellable hydrogel.

The expandable specimen can be expanded by contacting the swellablepolymer with a solvent or liquid to cause the swellable polymer toswell.

In one embodiment, prior to the treating step, the sample is subjectedto any suitable antigen retrieval process known to one of skill in theart and as further described below.

In one embodiment, the method comprises incubating the specimen withabout 1 to about 100 U/ml of a non-specific protease in a buffer havinga pH between about 4 and about 12, the buffer comprising about 5 mM toabout 100 mM of a metal ion chelator; about 0.1% to about 1.0% of anonionic surfactant; and about 0.05 M to about 1.0 M monovalent salt. Inone embodiment, the sample is incubated for about 0.5 to about 3 hoursat about 50° C. to about 70° C.

By “biomolecules” it is generally meant, but not limited to, proteins,lipids, steroids, nucleic acids, and sub-cellular structures within atissue or cell.

By “macromolecules” is meant proteins, nucleic acids, or small moleculesthat target biomolecules within the specimen. These macromolecules areused to detect biomolecules within the specimen and/or anchor thebiomolecules to the swellable polymer. For example, macromolecules maybe provided that promote the visualization of particular cellularbiomolecules, e.g., proteins, lipids, steroids, nucleic acids, etc. andsub-cellular structures. In some embodiments, the macromolecules arediagnostic. In some embodiments, the macromolecules are prognostic. Insome embodiments, the macromolecules are predictive of responsiveness toa therapy. In some embodiments, the macromolecules are candidate agentsin a screen, e.g., a screen for agents that will aid in the diagnosisand/or prognosis of disease, in the treatment of a disease, and thelike.

As used herein a bi-functional linker comprises reactive groups tofunctional groups (e.g., primary amines or sulfhydryls) on biomoleculeswithin the sample and a swellable hydrogel reactive group.

The bi-functional linker may be used to chemically modify the functionalgroup of biomolecules with a swellable hydrogel functional group, whichenables biomolecules within the sample to be directly anchored to, orincorporated into, the swellable hydrogel. In one embodiment, thebifunctional linker is a hetero-bifunctional linker. Hetero-bifunctionallinkers possess different reactive groups at either end of a spacer arm,i.e., atoms, spacers or linkers separating the reactive groups. Thesereagents not only allow for single-step conjugation of molecules thathave the respective target functional group, but they also allow forsequential (two-steps) conjugations that minimize undesirablepolymerization or self-conjugation. The bi-functional linker may be asmall molecule linker or a nucleic acid adaptor. In some embodiments thebifunctional linker is cleavable and can be referred to herein as acleavable crosslinker.

The anchor may be a physical, biological, or chemical moiety thatattaches or crosslinks the sample to the hydrogel. This may beaccomplished by crosslinking the anchor with the swellable hydrogel,such as during or after the polymerization, i.e., in situ formation ofthe swellable hydrogel. The anchor may comprise a polymerizable moiety.The anchor may include, but is not limited to, vinyl or vinyl monomerssuch as styrene and its derivatives (e.g., divinyl benzene), acrylamideand its derivatives, butadiene, acrylonitrile, vinyl acetate, oracrylates and acrylic acid derivatives. The polymerizable moiety may be,for example, an acrylamide modified moiety that may be covalently fixedwithin a swellable hydrogel.

As used herein, a “nucleic acid adaptor” is a nucleic acid sequencehaving a binding moiety capable of attaching to a nucleic acid and ananchor moiety capable of attaching to the swellable hydrogel. Attachingthe nucleic acid adaptor to a nucleic acid may be accomplished byhybridization or by ligation in situ. For example, DNA adaptors may beligated to the 3′ ends of the RNAs in the sample with RNA ligases, suchas T4 RNA ligase, or may be attached via a chemical linker such as areactive amine group capable of reacting with nucleic acid. Acrylamidemodified oligonucleotide primers may be covalently fixed within aswellable hydrogel such as a polyacrylate gel. As used herein, the term“acrylamide modified” in reference to an oligonucleotide means that theoligonucleotide has an acrylamide moiety attached to the 5′ end of themolecule.

As used herein, a “small molecule linker” is a small molecule having abinding moiety capable of attaching to a biomolecule within the sampleand an anchor moiety capable of attaching to the swellable hydrogel.Attaching the small molecule linker to the biomolecules may beaccomplished by hybridization or by a chemical reactive group capable ofcovalently binding. For example, LABEL-IT® Amine (MirusBio) is a smallmolecule with alkylating group that primarily reacts to the N₇ ofguanine, thereby allowing covalent binding of RNA and DNA. The smallmolecule linker may be, for example, acrylamide modified and thereforemay be covalently fixed within a swellable hydrogel. As used herein, theterm “acrylamide modified” in reference to a small molecule linker meansthat the small molecule linker has an acrylamide moiety.

In one embodiment, the bifunctional crosslinker comprises aprotein-reactive chemical moiety and a swellable hydrogel-reactivechemical moiety. The protein-reactive chemical group includes, but isnot limited to, N-hydroxysuccinimide (NHS) ester, thiol, amine,maleimide, imidoester, pyridyldithiol, hydrazide, phthalimide,diazirine, aryl azide, isocyanate, or carboxylic acid, which, forexample, can be reacted with amino or carboxylic acid groups on proteinsor peptides. In one embodiment, the protein-reactive groups include, butare not limited to, N-succinimidyl ester, pentafluorophenyl ester,carboxylic acid, or thiol. The gel-reactive groups include, but are notlimited to, vinyl or vinyl monomers such as styrene and its derivatives(e.g., divinyl benzene), acrylamide and its derivatives, butadiene,acrylonitrile, vinyl acetate, or acrylates and acrylic acid derivatives.

In one embodiment, the chemical to anchor proteins directly to anyswellable polymer is a succinimidyl ester of 6-((acryloyl)amino)hexanoicacid (acryloyl-X, SE; abbreviated “AcX”; Life Technologies). Treatmentwith AcX modifies amines on proteins with an acrylamide functionalgroup. The acrylamide functional groups allows for proteins to beanchored to the swellable polymer as it is synthesized in situ.

As used herein, the term “attach” or “attached” refers to both covalentinteractions and noncovalent interactions. In certain embodiments of theinvention, covalent attachment may be used, but generally all that isrequired is that the bi-functional linker remain attached to thebiomolecules. The term “attach” may be used interchangeably herein withthe terms, “anchor(ed)”, affix(ed), link(ed) and immobilize(d).

In certain embodiments, the biological sample can be labelled or taggedwith a detectable label. Typically, the label will bind chemically(e.g., covalently, hydrogen bonding or ionic bonding) to the sample, ora component thereof. The detectable label can be selective for aspecific target (e.g., a biomarker or class of molecule), as can beaccomplished with an antibody or other target specific binder. Thedetectable label preferably comprises a visible component, as is typicalof a dye or fluorescent molecule; however, any signaling means used bythe label is also contemplated. A fluorescently labeled biologicalsample, for example, is a biological sample labeled through techniquessuch as, but not limited to, immunofluorescence, immunohistochemical orimmunocytochemical staining to assist in microscopic analysis. Thus, thedetectable label is preferably chemically attached to the biologicalsample, or a targeted component thereof. In one embodiment, thedetectable label is an antibody and/or fluorescent dye wherein theantibody and/or fluorescent dye, further comprises a physical,biological, or chemical anchor or moiety that attaches or crosslinks thesample to the composition, hydrogel or other swellable material. In oneembodiment, the detectable label is attached to the nucleic acidadaptor. The labeled sample may furthermore include more than one label.For example, each label can have a particular or distinguishablefluorescent property, e.g., distinguishable excitation and emissionwavelengths. Further, each label can have a different target specificbinder that is selective for a specific and distinguishable target in,or component of the sample.

The biological sample can be anchored to a swellable hydrogel beforeexpansion. The anchoring can be accomplished by anchoring thebifunctional crosslinker with the swellable hydrogel, such as during orafter the polymerization or in situ formation of the swellable hydrogel.

In some embodiments, the bifunctional crosslinker is attached to the Xor X₁ moiety of the monomer of Formula (A), Y₁ of the monomer of Formula(B1), or Y₂ of the monomer of Formula (B2). The bifunctional crosslinkermay comprise a small molecule linker capable of attaching to thebiological sample and to the hydrogel. Examples of small moleculelinkers include NHS-azide and NHS-DBCO which can react with the arms ofthe monomers of the hydrogel. For example, a protein and/or an antibodycan be anchored to the hydrogel with NHS-azide or DBCO-NHS. In the caseof NHS-azide, the NHS moiety binds to the label and the azide reactswith an alkyne group in the hydrogel (e.g., Y₁ or Y₂ of Formulae (B1)and (B2)) (for example, DBCO) by click chemistry. In the case ofNHS-DBCO, the NHS moiety binds to the label and the DBCO reacts with anazide (e.g., X of Formula (A)) in the hydrogel by click chemistry.

FIG. 5 demonstrates an implementation of such anchoring. Yellowfluorescent proteins (YFPs) in a Thy1-YFP mouse brain slice are retainedby a small-molecular linker, NHS-azide. The YFP molecules were firstreacted with NHS-azide and then anchored to the hydrogel byclick-reaction. The gel was expanded and immunostained by antibodies(anti-GFP) post-expansion for visualization. FIG. 6 shows expansion ofantibody-stained HEK cells using the tetragel. The dye-conjugatedsecondary antibodies are anchored to the tetragel by NHS-azide. Othermolecules such as RNAs and lipids, for example, can be similarlyanchored to the gel through similarly designed small-molecule linkers.

In another implementation, both GFP and Cy3-conjugated secondaryantibodies are linked into the tetragel using NHS-azide. FIG. 7 showspre- and post-expansion images of nuclear pore complex with simultaneousGFP labeling and antibody staining.

In some embodiments, after the sample has been anchored to the swellablehydrogel, the sample is, optionally, subjected to a disruption of theendogenous biological molecules or the physical structure of thebiological sample, leaving the linkers intact and anchored to theswellable material. In this way, the mechanical properties of thesample-swellable material complex are rendered more spatially uniform,allowing isotropic expansion with minimal artifacts.

As used herein, the “disruption of the endogenous physical structure ofthe sample” or the “disruption of the endogenous biological molecules”of the biological sample generally refers to the mechanical, physical,chemical, biochemical or, enzymatic digestion, disruption or break up ofthe sample so that it will not resist expansion. In one embodiment, aprotease enzyme is used to homogenize the sample-hydrogel complex. It ispreferable that the disruption does not impact the structure of thehydrogel but disrupts the structure of the sample. Thus, the sampledisruption should be substantially inert to the hydrogel. The degree ofdigestion can be sufficient to compromise the integrity of themechanical structure of the sample or it can be complete to the extentthat the sample-hydrogel complex is rendered substantially free of thesample. In one embodiment, the disruption of the physical structure ofthe sample is protein digestion of the proteins contained in thebiological sample. The sample-hydrogel complex is then isoptropicallyexpanded. In one embodiment, a solvent or liquid is added to the complexwhich is then absorbed by the hydrogel and causes swelling. Where thehydrogel is water swellable, an aqueous solution can be used.

As described herein, the expanded and/or labelled sample can be viewedusing microscopy. The sample can be imaged using an optical microscope,allowing effective imaging of features below the classical diffractionlimit. Where the resultant specimen is transparent, custom microscopescapable of large volume, widefield of view, 3D scanning can also be usedin conjunction with the expanded sample.

In one embodiment, the addition of water an aqueous solution allows forthe embedded sample to expand relative to its original size inthree-dimensions. Thus, the sample can be increased 100-fold or more involume. This is because the polymer is embedded throughout the sample,therefore, as the polymer swells (grows) it expands the tissue as well.Thus, the tissue sample itself becomes bigger. As the material swellsisotropically, the anchored tags maintain their relative spatialrelationship.

Use of the tetragel system, as described here, for ExM may eliminateradicals during the in situ polymerization. In radical polymerization,radical species are known to damage organic molecules such asfluorescent dyes and tags in biological samples (e.g., molecules thathave C—C double bonds). As described, tetragels are formed by linkingmonomers with a non-radical, bio-orthogonal reaction. Therefore, thehydrogel formation introduces much less chemical damage to biomoleculesand tags, reserving the chemical structures as well as increasing theretention after expansion. For example, the C—C double bonds present insome of the far-red dyes can be robustly retained after gelation andexpansion with tetragel (FIG. 8 ).

In addition to a single round of expansion, the hydrogels describedherein can be used in the iterative ExM (iExM) process. The process ofiteratively expanding the samples can be applied to samples that havebeen already expanded using the techniques described herein one or moreadditional times to iteratively expand them such that, for example, a5-fold expanded sample can be expanded again 3- to 4-fold, resulting inas much as a 17- to 19-fold or more linear expansion. The iExM procedurebegins with first conducting ExM on a sample and further provides one ormore additional and iterative expansions of the sample by forming, forexample, another hydrogel inside an expanded hydrogel such as the firstexpanded hydrogel of the ExM method. The iterative ExM methods of thepresent claims comprising using the hydrogel described herein (formed bythe reaction of a monomer of Formula (A) with a monomer of Formula (B1)or (B2) for any steps including the first gel, the second gel, or bothfirst and the second gel.

In one embodiment, in iExM, the first swellable material and thenon-swelling material are made with a different crosslinker compared tothe second swellable material in order to selectively digest the firstswellable material and the non-swellable re-embedding material while thesecond swellable material remains intact. Selective digestions of eachsuccessive swellable material depends on the conditions under which thecross-linkers of the target swellable material cleavable. For example,swellable materials crosslinked with DHEBA, may be cleaved and dissolvedby treatment with 0.2M sodium hydroxide for 1 hour. Swellable materialsmade with BAC can be dissolved and the crosslinker cleaved by treatmentwith Tris(2-carboxyethyl)phosphine hydrochloride (TCEP).

In some embodiments, the method comprises:

-   -   a) permeating a biological sample with a first hydrogel, wherein        the sample is anchored to the hydrogel;    -   b) swelling the first hydrogel resulting in a first expanded        sample;    -   c) permeating the first expanded sample with a second hydrogel;        and    -   d) swelling the second hydrogel resulting in a second expanded        sample;        wherein the first hydrogel and/or the second hydrogel is the        product a non-radical polymerization reaction between a monomer        of Formula (A) and a monomer of Formula (B1) or (B2).

In some embodiments, the invention provides method for enlarging asample of interest for microscopy, the method comprising the steps of:

-   -   a) embedding a sample in a first hydrogel;    -   b) swelling the first hydrogel to form a first enlarged sample;    -   c) re-embedding the first enlarged sample in a non-swellable        material;    -   d) embedding the first enlarged sample in a second hydrogel; and    -   e) swelling the second hydrogel to form a second enlarged sample        that is enlarged as compared to the first enlarged sample;        wherein the first hydrogel and/or the second hydrogel is the        product a non-radical polymerization reaction between a monomer        of Formula (A) and a monomer of Formula (B1) or (B2).

The term “re-embedding” comprises permeating (such as, perfusing,infusing, soaking, adding or other intermixing) the sample with aswellable or non-swellable material, preferably by adding precursorsthereof. Alternatively or additionally, embedding the sample in anon-swellable material comprises permeating one or more monomers orother precursors throughout the sample and polymerizing the monomers orprecursors to form the non-swellable material or polymer. In thismanner, the first enlarged sample, for example, is embedded in thenon-swellable material. Embedding the expanded sample in a non-swellablematerial prevents conformational changes during sequencing despite saltconcentration variation. The non-swellable material can becharge-neutral hydrogels. For example, it can be polyacrylamidehydrogel, composed of acrylamide monomers, bisacrylamide crosslinker,ammonium persulfate (APS) initiator and tetramethylethylenediamine(TEMED) accelerator.

In one embodiment, the cleavable version of the tetragel (e.g., MonomerA in FIG. 2B; Monomer B in FIG. 3A) can be used as the first gel foriExM. After gelation, the cleavable tetragel can be then reembedded,gelled and then cleaved for the 2^(nd) round of expansion. In this iExMprocess, the linker may have a functional group that can be anchored tothe tetragel, such as a DBCO or azide group.

In one embodiment, the biological sample and each enlarged samplethereafter is permeated with one or more monomers or a solutioncomprising one or more monomers or precursors which are then reacted toform a swellable or non-swellable polymerized gel depending on what stepof the method is being performed. For example, if the biological sampleis to be embedded in hydrogel AB2, a solution comprising the monomer ofFormula (A) and a monomer of Formula (B2) can be perfused throughout thesample.

The invention also includes a method for preparing and amplifyingnucleic acids in situ and methods for in-situ sequencing of targetnucleic acids in an expanded composite comprising biological sample anda hydrogel as described herein. Methods of preparing and amplifyingnucleic acids and sequencing using expanded or enlarged composites(“expansion sequencing” or “ExSEQ”) have been described, for example, inU.S. Pat. App. Pub. No. 2016/0304952 and U.S. patent application Ser.No. 15/789,419, the contents of which are expressly incorporated byreference herein. Expanding specimens before sequencing separatessequencing targets by a programmable volumetric factor, enablingdetection of multiple species within a diffraction-limited spot in apre-expansion space, using diffraction limited microscopy in apost-expansion space. In addition, expansion results in homogenizationof the chemical environment (which is ˜99% water throughout the specimenin the expanded state) and providing optical clarity. In certainaspects, the method comprises the steps of:

-   -   a. attaching target nucleic acids present in the sample with a        bifunctional crosslinker;    -   b. permeating the sample with a monomer of Formula (A) and a        monomer of Formula (B1) or (B2) under conditions suitable to        form a hydrogel by non-radical polymerization and thereby        forming a sample—hydrogel complex, wherein the bifunctional        crosslinker is attached both to the target nucleic acids present        in the sample and to the hydrogel;    -   c. digesting proteins present in the sample; and    -   d. expanding the complex to form a first enlarged composite.

In one embodiment, the method further comprises re-embedding the firstenlarged sample in a non-swellable material to form a re-embeddedsample.

In one embodiment, the method further comprises modifying the targetnucleic acids or the nucleic acid adaptor to form a target nucleic acidsor a nucleic acid adaptor useful for sequencing. In this manner, thenucleic acids present in the re-embedded composite may be sequenced.

“Modifying the target nucleic acids or the nucleic acid adapter” can,for example, refer to biochemical modification, for example, contactingthe target nucleic acids or the nucleic acid adapter with reversetranscriptase. In certain examples, the nucleic acid adaptors areattached to target nucleic acids via ligation to the target nucleicacid. In one embodiment, the nucleic acid adaptors are attached totarget nucleic acids via a chemical reagent capable of reacting withamine groups on the target nucleic acid.

The term “nucleic acid” and “polynucleotide” are used interchangeablyherein to refer to a polymer having multiple nucleotide monomers. Anucleic acid can be single- or double-stranded, and can be DNA (e.g.,cDNA or genomic DNA), RNA, or hybrid polymers (e.g., DNA/RNA). Nucleicacids can be chemically or biochemically modified and/or can containnon-natural or derivatized nucleotide bases. “Nucleic acid” does notrefer to any particular length of polymer e.g., greater than about 2bases, greater than about 10 bases, greater than about 100 bases,greater than about 500 bases, greater than 1000 bases, greater than10,000 bases, greater than 100,000 bases, greater than about 1,000,000,up to about 10¹⁰ or more bases composed of nucleotides. Additionally, apolynucleotide can be native to the sample (for example, present in thesample at the time the sample is obtained from the original organism).Alternatively, a polynucleotide can be artificial or synthetic, such aswhen the polynucleotide is added to the sample to cause hybridization toa target nucleic acid. The term “polynucleotide” is intended to includepolynucleotides comprising naturally occurring nucleotides and/ornon-naturally occurring nucleotides. Non-naturally occurring nucleotidescan include chemical modifications of natural nucleotides. In this case,it is preferred that the synthetic polynucleotides can hybridize to thetagged genomic fragments.

The term “sequence,” in reference to a nucleic acid, refers to acontiguous series of nucleotides that are joined by covalent bonds(e.g., phosphodiester bonds).

The term “target nucleic acid” refers to a nucleic acid whose presencein a sample may be identified and sequenced. A target nucleic acid canbe any nucleic to be selected and, optionally, amplified or sequencedpreferably in combination with the nucleic acid adaptor. Target nucleicacids for use in the provided methods may be obtained from anybiological sample using known, routine methods.

In one embodiment, the method further comprises the step of passivatingthe first swellable material. As used herein the term “gel passivation”refers to the process for rendering a gel less reactive with thecomponents contained within the gel such as by functionalizing the gelwith chemical reagents to neutralize charges within the gel. Forexample, the carboxylic groups of sodium acrylate, which may be used inthe swellable gel, can inhibit downstream enzymatic reactions. Treatingthe swellable gel composed of sodium acrylate with1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) andN-Hydroxysuccinimide (NHS) allows primary amines to covalently bind thecarboxylic groups to form charge neutral amides and passivate theswellable gel. After re-embedding in the non-swellable gel, theswellable gel may also be partially or completely degraded chemically,provided that the target nucleic acids can either stay anchored or canbe transferred to the non-swellable gel.

As described above, the biological sample can be labelled or tagged, forexample, with a detectable label. Also as described above, thebiological sample can be treated with a detergent prior to beingcontacted with the hydrogel precursor(s).

In accordance with the invention, chemically fixed and permeabilizedbiological specimens are expanded. The expanded gel may be converted toa non-expanding state, by re-embedding in a non-expanding material. RNAor DNA molecules present in the sample may be sequenced using methodsknown to those familiar with the art, including sequencing byhybridization, ligation, and synthesis. Sequencing can be carried out byany method known in the art including, but not limited to, sequencing byhybridization, sequencing by ligation or sequencing by synthesis.General sequencing methods known in the art, such as sequencing byextension with reversible terminators, fluorescent in situ sequencing(FISSEQ), pyrosequencing, massively parallel signature sequencing (MPSS)and the like are suitable for use in the methods of the invention.Reversible termination methods use step-wise sequencing-by-synthesisbiochemistry that coupled with reversible termination and removablefluorescence.

FISSEQ is a method whereby DNA is extended by adding a single type offluorescently labelled nucleotide triphosphate to the reaction; washingaway unincorporated nucleotide, detecting incorporation of thenucleotide by measuring fluorescence, and repeating the cycle. At eachcycle, the fluorescence from previous cycles is bleached or digitallysubtracted or the fluorophore is cleaved from the nucleotide and washedaway. FISSEQ is described, for example in, (Lee et al., Science. 343,1360-3 (2014).

Pyrosequencing is a method in which the pyrophosphate (PPi) releasedduring each nucleotide incorporation event (i.e., when a nucleotide isadded to a growing polynucleotide sequence). The PPi released in the DNApolymerase-catalyzed reaction is detected by ATP sulfurylase andluciferase in a coupled reaction which can be visibly detected. Theadded nucleotides are continuously degraded by a nucleotide-degradingenzyme. After the first added nucleotide has been degraded, the nextnucleotide can be added. As this procedure is repeated, longer stretchesof the template sequence are deduced. Pyrosequencing is describedfurther in Ronaghi et al. (1998) Science 281:363.

MPSS utilizes ligation-based DNA sequencing simultaneously. A mixture oflabelled adaptors comprising all possible overhangs is annealed to atarget sequence of four nucleotides. The label is detected uponsuccessful ligation of an adaptor. A restriction enzyme is then used tocleave the DNA template to expose the next four bases. MPSS is describedfurther in Brenner et al., (2000) Nat. Biotech. 18: 630.

The invention is illustrated by the following examples which are notmeant to be limiting in any way.

EXEMPLIFICATION

Materials and Methods

I. Synthesis of Monomer A

Synthesis of Br-Terminated Monomer (1):

Synthesis of (1) was carried out with a modified procedure of asdescribed in Oshima et al. Before use, t-butyl acrylate was purifiedwith an inhibitor removal column to remove trace amounts of4-methoxyphenol. To 640 mg of CuBr and 48 mg of CuBr₂ was added 128 mLof t-butyl acrylate, and the mixture was bubbled with dry nitrogen at50° C. A total of 1.03 mL of PMDETA was then added dropwise. After 5-10min, a solution of 1.6 g of star core (pentaerythritoltetrakis(2-bromoisobutyrate)) dissolved in 16 mL acetone was addeddropwise. The reaction was carried out for 90 min at 50° C., with drynitrogen bubbling for the first ˜10 min. Unreacted t-butyl acrylate wasthen removed by rotary evaporation. The crude product mix was thendissolved in DMF and precipitated with water. Precipitation was repeatedan additional two times, yielding 15.3 g of a white powder.

Synthesis of N₃-Terminated Monomer (2):

A total of 15.3 g of Br-terminated monomer (1) was dissolved in 80 mL ofDMF. Excess sodium azide (exceeding its solubility limit in DMF) wasadded to the mixture, and the reaction was carried out overnight at roomtemperature. The supernatant was subsequently decanted and precipitatedwith water, yielding 11 g of a white powder.

Synthesis of Deprotected Monomer (3):

A total of 5.04 g of (2) was dissolved in 30 mL of CH₂Cl₂, followed byaddition of 15 mL of trifluoroacetic acid. The reaction was carried outat 4° C., resulting the gradual precipitation of a white powder. After24-48 h, the product was collected by centrifugation, washed withacetone, and dried in a low-humidity chamber. The product wasre-suspended in a solution of NaOH/H₂O to a final concentration of 200mg/mL and pH 7.

Monomer (3) is referred to as “monomer 1” in the section below.

2. Synthesis of Monomer B

Amine-terminated PEG monomer (10 kDa; purchased from NOF) was dissolvedin DMSO to a concentration of 100-200 mg/mL. BCN—NHS, DBCO—NHS orDBCO-sulfo-NHS (referred to as Monomer B as well as monomers 2, 2′, and2″, respectively, below) (1:1 molar ratio to amines) was then added tothe solution. The conjugation reaction was carried out overnight. Theproduct solution was used for gelation reactions without furtherwork-up.

3. Gelation

Cultured cells and brain slices were fixed and immunostained aspreviously described in Tillberg et. al. Unless otherwise noted,single-round expansion of cells and tissues were carried out using thesubsequent standard gelation protocol. Fixed biological samples weresoaked in 0.1 mg/mL NHS-azide in 1× PBS overnight and washed with 1× PBStwice immediately before the gelation.

Monomer A and Monomer B solutions were mixed with a 1:1 molar ratio andadditional water was added to a final concentration of ˜3.3 wt % forMonomer A to yield the gelling solution. In a typical implementation, 10uL of Monomer A (200 mg/mL), 10 uL of Monomer B (200 mg/mL), and 40 ulof water were mixed and vortexed to give the gelling solution. Thesamples were gelled in the gelling solution at 4° C. for 8 hours. Forexample, 10 μL of monomer 1 (200 mg/mL), 10 μL of monomer 2 (200 mg/mL),and 40 μl of water were mixed. After applying the gelling solution tothe samples in the gelation chamber as previously described, gelationwas carried out overnight at 4° C. The amount of monomers 1 and 2 (or2′, 2″) and the mixed gelling solution was scaled up and downproportionally according to the size and number of the samples.

4. Digestion and Expansion

Gelled samples were digested overnight in digestion buffer withproteinase K (8 units/mL) as described in Tillberg, et. al. The sampleswere washed in excessive amount of water 3 times for 20 min each timefor the expansion.

For fluorescein visualization of tetragels (TGs) (FIGS. 4-6 and 8-9 ), atrace amount of fluorescein amine was mixed in the gelling solutionbefore gelation for 1-2 hours at 37° C. Briefly, a stock solution of ˜50mM fluorescein-azide was prepared by adding 5 μL of 100 mMfluorescein-amine in DMSO to 5 μL of 20 mg/mL NHS-azide in DMSO. ˜3 μLof the fluorescein-azide stock solution was then added to ˜60 μL of thegelling solution (with monomers 2 or 2′ or 2″) before gelation incircular molds of ˜3 mm diameters.

5. Imaging

All samples were imaged with an Andor spinning disk (CSU-W1 Yokogawa)confocal system on a Nikon Eclipse Ti-E microscope body or a NikonEclipse Ti-E widefield microscope. High resolution images were collectedon the spinning disk confocal system using a CFI Apo LambdaS LWD 40×,1.15 NA water-immersion objective.

6. HeLa Cell Culture. HeLa Cell Expansion (Post-Expansion Staining andMulti-Round Iterative Expansion).

High-Temperature Treatment for Post-Gelation Antibody Staining

After gelation, sample chambers were gently opened with a razor blade.Empty gels surrounding the circular coverslips containing HeLa cultureswere trimmed away. Circular coverslips were placed into autoclave-safeglass vials containing 1 mL of 1× PBS+1M NaCl, and incubated at RT for30 min. Samples were incubated in MAP Buffer (200 mM SDS+200 mM NaCl+50mM Tris, pH to 9.0) overnight at 37° C., followed by a 3 hr incubationat 70° C. and 1 hr incubation at 95° C. Following the high-temperaturetreatment, samples were cooled to RT and washed 4 times in PBST (1×PBS+0.1% Triton X-100) for 30 min each. Samples were stainedsequentially with sheep anti-tubulin primary antibody and anti-sheepsecondary antibody. Each staining step was performed in PBST at RT withovernight incubation, followed by 3 washes in PBST for 1 hr each.

7. HSV-1 Virion Expansion (Direct Labeling and Multi-Round IterativeExpansion)

Immobilization and Fixation

Purified HSV-1 virion stock was prepared by the Viral Core Facility atthe Massachusetts General Hospital (MGH) as previously described. TheHSV stock was diluted to a functional titer of 2.5×10⁸ functionalvirions/mL in PBS and kept on ice until immobilization. A #0 circular12-mm coverslip was treated with oxygen plasma for 1 min. Immediatelyafter the treatment, 30 uL of the diluted HSV-1 solution was drop-castedto the coverslip and incubated for 15 min at room temperature. Theimmobilized viruses were fixed in PBS+4% PFA for 10 min, and then washedwith PBS twice, each time for 5 min.

Oligo Conjugation to Envelope Proteins

Envelope proteins on the fixed viruses were conjugated to a DNA oligowith the SoluLink bioconjugation chemistry as previously described. Theoligo provided a molecular handle for signal anchoring, transfer andamplification through the iterative expansion process. Briefly, a 19-bpoligo (sequence B1′) with a 5′ amine modification (Integrated DNATechnologies) was purified with ethanol precipitation and reacted withSulfo-S-4FB overnight in Buffer A (150 mM NaCl, 100 mM Na₂HPO₄, pH 7.4)at a molar ratio of 1:15. The S4FB-reacted oligo was purified with asize exclusion filter, and then stored at 4° C. Fixed virusesimmobilized on the coverslip were washed in Buffer A for 5 min, and thenincubated with 100 uL of 160 mM of S-HyNic in Buffer A for 2 hours atroom temperature. The S-HyNic-reacted viruses were washed with Buffer C(150 mM NaCl, 100 mM Na₂HPO₄, pH 6.0) twice, each time for 5 min. Oligoconjugation solution was prepared by first adding 50 nmol of purifiedS4FB-reacted oligo to 100 uL of Buffer C, and then adding an amount of10× TurboLink Catalyst Buffer that equals to 1/9 of the combined volume.The S-HyNic-reacted viruses were incubated in the oligo conjugationbuffer overnight at room temperature in a humidified chamber. Finally,the oligo-conjugated viruses were washed 3 times with PBS, each time for10 min, and then incubated in detergent-free hybridization buffer (10%Dextran sulfate, 1 mg/mL yeast tRNA, 5% NDS, 2×SSC) for 3 hours at roomtemperature. Viruses were incubated with 4 nmol of oligo B1-acrydite oroligo B1-azide (for PAA or TG, respectively) in 300 uL of detergent-freehybridization buffer, overnight at room temperature, and then washed 3times in PBS, each time for 10 min.

Gelation and Digestion

As described in the previous section, cleavable TG gelling solution wasprepared by mixing monomer 1 (200 mg/mL) and monomer 2″ (200 mg/mL) at amolar ratio of ˜1:1 and adding water to adjust the final concentrationof monomer 1 to ˜3.3% (w/v). BAC-crosslinked cleavable PAA gellingsolution was prepared as previously described. A gelation chamber wasconstructed around the virus-immobilized coverslip by the followingsteps. First, the coverslip was transferred to the center of a glassslide. Spacers consisting of a stack of a #0 and a #1 coverslip wereplaced on either side of the virus-immobilized coverslip. 50 uL offreshly prepared TG or PAA gelling solution was added to the coverslip,and the chamber was closed by placing a rectangular coverslip on top ofthe spacers. The gelling solution was further added from the side of thechamber until the chamber was completely filled. The gelation chamberswere incubated for 2 hours at 37° C. After gelation, the chambers werepartially opened using a diamond scribe to remove portions of the topcover glass that were not directly above the virus-immobilizedcoverslip. The chambers were then placed into a rectangular 4-well dishand incubated in digestion buffer with Proteinase K at 8 U/mL (NewEngland Bio Labs; 1:100 dilution) overnight at room temperature withgentle shaking. After digestion, the top cover glass was removed. Thediameter of the indentation casted by the circular 12-mm coverslip wasmeasured for downstream estimation of the overall expansion factor.Regions inside of the circular 12-mm coverslip (i.e. regions with theimmobilized viruses) were trimmed into a parallelogram, whose shape canbe used to indicate the side of the gel that the viruses were located.The side lengths of the parallelogram were measured. Finally, trimmedgels were washed twice in PBS, each time for 10 min. To de-hybridize B1′and B1 oligos, the gels were incubated in 80% formamide at roomtemperature with gentle shaking, and then washed 3 times in PBS, eachtime for 10 min.

Re-Embedding Into a BAC-Crosslinked Non-Expanding 2^(nd) Gel

The gels were transferred (with the virus side down, as deduced from theshape of the parallelogram) into a rectangular 4-well dish that carriesa glass slide in each well, and expanded in water 3 times, each time for30 min. Gels were then incubated in 3 mL of BAC non-expanding gellingsolution (10% acrylamide, 0.2% BAC, 0.05% TEMED, 0.05% APS) for 5 minwith gentle shaking. After the incubation, the non-expanding gellingsolution was removed from the well, and the glass slide carrying theexpanded gel was transferred to a gelation chamber. Spaces consisting ofa stack of #1.5 cover glasses were placed on either side of the gel, andthe chamber was closed with a rectangular cover glass. The non-expandinggelling solution was added from the side of the chamber until thechamber was completely filled. The gelation chambers were incubated for2 hours at 37° C. After gelation, the chambers were opened by removingthe top cover glass. Side lengths of the parallelogram were measured.The gels were trimmed to leave only the portion inside theparallelogram, while preserving the shape of the parallelogram. Sidelengths of the trimmed gels were measured. Finally, the trimmed gelswere washed twice in PBS, each time for 10 min.

1^(st) Linker Hybridization

The gels were incubated in hybridization buffer (4×SSC+20% formamide)for 30 min at room temperature. For readout after 2-round expansion(˜10-20× expansion factor), the gels were incubated with 1 nmol of oligo5′Ac-B1′-4xB2′ in 500 uL of hybridization buffer overnight at roomtemperature. For readout after 3-round expansion (˜40-80× expansionfactor), the gels were incubated with 1 nmol of oligo 5′Ac-B1′-A2′ in500 uL of hybridization buffer overnight at room temperature. Afterincubation, the gels were washed in hybridization buffer 3 times, eachtime for 1 hour, and then overnight, all with gentle shaking. The gelswere then washed 3 times in PBS, each time for 5 min.

Re-Embedding Into a DATD-Crosslinked Expanding 3^(rd) Gel

The gels were incubated in DATD expanding gelling solution (7.5% sodiumacrylate, 2.5% acrylamide, 0.5% DATD, PBS, 2M NaCl, 0.01% 4-HT, 0.2%TEMED, 0.2% APS) for 30 min at 4° C. The gels (with the virus side down)were enclosed in gelation chambers, incubated for 2 hours at 37° C.,size-measured, trimmed, size-re-measured, and washed as described in“Re-embedding into a BAC-crosslinked non-expanding 2^(nd) gel”.

Cleaving BAC-Crosslinked 1^(st and) 2^(nd) Gels

The gels were incubated in BAC-cleaving buffer (0.25M TCEP-HCl, 0.75MTris-HCl, pH 8.0) overnight at room temperature. The gels were thenwashed 4 times in PBS, each time for 30 min. For samples designated for3-round expansion, the gels were incubated in thiol-blocking buffer (100mM maleimide, 100 mM MOPS, pH 7.0) for 2 hours at room temperature. Thethiol-blocked gels were washed 3 times in PBS, each time for 10 min.

LNA Hybridization for Readout After 2-Round Expansion

For samples designated for 2-round expansion, the gels were incubatedwith 1 nmol of LNA_B2-Atto647N in 500 uL of hybridization buffer. TheLNA-hybridized gels were washed in hybridization buffer 3 times, eachtime for 1 hour, and then overnight, all with gentle shaking. The gelswere then washed 3 times in PBS, each time for 5 min.

Gel Expansion, Immobilization, and Imaging for 2-Round Expanded Samples

The gels were trimmed into smaller pieces (˜5×5 mm) while preserving theshape of the parallelogram. First, the gels were transferred (with thevirus side down, as deduced from the shape of the parallelogram) into arectangular 4-well dish that carries a glass slide in each well, andexpanded in water 3 times, each time for 30 min. Wells in a glass-bottom6-well plate were modified with poly-lysine as previously described. Theexpanded gels were then gently transferred to the poly-lysine modifiedglass-bottom plate for imaging as previously described.

Re-Embedding into a DATD-Crosslinked Non-Expanding 4^(th) Gel (for3-round expansion)

Thiol-blocked gels in “Cleaving BAC-crosslinked 1^(st) and 2^(nd) gels”were subsequently trimmed into smaller pieces (˜5×5 mm) while preservingthe shape of the parallelogram. The gels were transferred (with thevirus side down, as deduced from the shape of the parallelogram) into arectangular 4-well dish that carries a glass slide in each well, andexpanded in water 3 times, each time for 30 min. The gels weretransferred onto a slide glass and trimmed in the z-direction into athickness of 1 mm. Briefly, the glass slide (with 1-mm thickness)carrying the expanded sample was placed between two stacks of 1-mm-glassslides, and a cryostat blade was pushed slowly through the expanded gel.The bottom gel, which carries the virus at the bottom side, wastransferred back to the 4-well plate. The z-trimmed gels were thenincubated in DATD non-expanding gelling solution (10% acrylamide, 0.5%DATD, 0.05% TEMED, 0.05% APS) for 30 min at 4° C. The gels were enclosedin gelation chambers, incubated for 2 hours at 37° C., size-measured,trimmed, size-re-measured, and washed as described in “Re-embedding intoa BAC-crosslinked non-expanding 2^(nd) gel”.

2^(nd) Linker Hybridization

The gels were incubated in hybridization buffer (4×SSC+20% formamide)for 30 min at room temperature. The gels were incubated with 0.5 nmol ofoligo 5′Ac-A2-4xB2′ in 1 mL of hybridization buffer overnight at roomtemperature. After incubation, gels were washed in hybridization buffer3 times, each time for 1 hour, and then overnight, all with gentleshaking. The gels were then washed 2 times in PBS, each time for 30 min.

Re-Embedding Into a Bis-Crosslinked Expanding 5^(th) Gel

The gels were incubated in bis expanding gelling solution (7.5% sodiumacrylate, 2.5% acrylamide, 0.15% bis, PBS, 2M NaCl, 0.01% 4-HT, 0.2%TEMED, 0.2% APS) for 30 min at 4° C. The gels (with the virus side down)were enclosed in gelation chambers, incubated for 2 hours at 37° C.,size-measured, trimmed, size-re-measured, and washed in the same way asdescribed in “Re-embedding into a BAC-crosslinked non-expanding 2^(nd)gel”.

Cleaving DATD-Crosslinked 4^(th) and 5^(th) Gels

The gels were incubated in DATD-cleaving buffer (20 mM sodium periodate,PBS, pH 5.5) for 30 min at room temperature. The gels were then washed 3times in PBS, each time for 30 min, and then overnight with gentleshaking.

LNA Hybridization for Readout After 3-Round Expansion

The gels were hybridized with LNA_B1_Atto647N as described in “LNAhybridization for readout after 2-round expansion”.

Gel Expansion, Immobilization, and Imaging

The gels were trimmed, expanded, immobilized and imaged as described in“Gel expansion, immobilization, and imaging for 2-round expandedsamples”.

Expansion Factor Estimation

Side lengths of the gels were recorded before and after each trimmingstep (for example, after every re-embedding step and before everyimmobilization step) and immediately before imaging. Single-stageexpansion factor was calculated by taking the average quotient betweenthe pre-trimming size of the current step and the post-trimming size ofthe previous step. Overall expansion factor was calculated from theproduct of all the previous single-step expansion factors till aspecific step.

HSV-1 Virion Diameter Analysis.

Diameters of the HSV-1 virion envelope protein layer were measured witha semi-automated analysis pipeline implemented on MATLAB (“ParticleAnalysis Assistant”). The MATLAB code for “Particle Analysis Assistant”is available for download. Briefly, within an acquired image z-stack,all round objects with a local minimum inside the object were identifiedas the virions and were analyzed. First, the center of each virion wasdetermined manually within the image z-slice that had the largest viriondiameter. Next, the line profile of the virion was automaticallymeasured across the virion center along the x-axis and the distancebetween the two local maxima in the line profile was recorded as thevirion diameter (along the x-axis). When the automated diametermeasurement failed due to fluorescent signals from adjacent virions orunspecific fluorescent label readouts, manual correction of the peaklocations was performed. Virions with repeated measurement failures weremanually rejected from the final statistics. Student's two-tailed t-testwas used to determine the statistical significance between the meandiameters derived from the TG- and PAA-based iteratively expanded HSV-1virions.

Averaged Single Particle Images of HSV-1 Virions.

Single HSV-1 virion particle images were generated using asemi-automated analysis pipeline implemented on MATLAB (“ParticleAnalysis Assistant”). First, the center of the virions in the previousdiameter analysis was inspected and re-aligned manually. During theinspection, a small portion (<10%) of the virions, which had significantoverlaps with the neighboring virions, were rejected for averaging.Next, the single virion image around each virion center wasautomatically cropped, calibrated with the expansion factor, and thenaveraged.

TABLE 1 DNA oligo sequences. Oligo Name Purpose Sequence (IDT format)SEQ ID NO Modification 5′Amine-B1′ Pre-G1 conjugationAAT ACG CCC TAA GAA TCC GAA 1 5′ Amino to envelope proteins CModifier C6 5′Acrydite- Pre-G1 adaptor for GTT CGG ATT CTT AGG GCG TA 25′ Acrydite B1 PAA-based iExM 3′Azide-B1 Pre-G1 adaptor forGTT CGG ATT CTT AGG GCG TA 3 3′ Azide TG-based iExM 5′Acrydite-Post-G2 linker for 2- TAC GCC CTA AGA ATC CGA ACA 4 5′ AcryditeB1′-4xB2′ round iExM TGC ATT ACA GCC CTC AAT GCATTA CAG CCC TCA ATG CAT TAC AGC CCT CAA TGC ATT ACA GCC CTC A5′Acrydite- Post-G2 linker for 3- TAC GCC CTA AGA ATC CGA ACA 55′ Acrydite B1′-A2′ round iExM TGG TGA CAG GCA TCT CAA TCT 5′Acrydite-Post-G4 linker for 3- AGA TTG AGA TGC CTG TCA CCA 6 5′ Acrydite A2-4xB2′round iExM TGC ATT ACA GCC CTC AAT GCA TTA CAG CCC TCA ATG CAT TACAGC CCT CAA TGC ATT ACA GCC CTC A LNA_B2- Post-G3 or Post-G5TGAGGGCTGTAATGC 7 3′ Atto Atto647N readout 647N, LNAs (underlined)

TABLE 2 Recipes of hydrogel gelling solutions. Gel Name Purpose RecipeTG System Monomer 2″ Monomer H₂O (200 mg/mL) 1 (200 mL/mL)Cleavable tetra-gel 1^(st) Gel for TG- 2 parts 1 part 3 parts based iExMPAA System Sodium Cross- Acrylamide acrylate linker PBS NaClBAC-crosslinked 1^(st) Gel for  2.5% 7.5% 0.2% BAC 1x 2M expanding gelPAA-based iExM BAC-crosslinked 2^(nd) Gel 10% 0 0.2% BAC 0 0non-expanding gel DATD-crosslinked 3^(rd) Gel  2.5% 7.5% 0.5% 1x 2Mexpanding gel DATD DATD-crosslinked 4^(th) Gel 10% 0 0.5% 0 0non-expanding gel DATD Bis-crosslinked 5^(th) Gel  2.5% 7.5% 0.15% bis1x 2M expanding gelResults and Discussion

To test if the synthesized monomers form hydrogels, we first mixed astoichiometrically equal amount of monomer 1 and monomer 2 (or 2 or 2″)and casted the monomer solution to a circular mold (as discussed above).It was found that, the monomer mixtures indeed formed hydrogels that areoptically transparent and mechanically elastic after a 1- to-2-hourincubation at 37° C. To track the overall shapes and sizes of the gels,we mixed to the monomer solutions a trace amount of fluorescent dyes,and imaged the gels using fluorescence microscopy. Similar to radicallypolymerized polyacrylamide/sodium polyacrylate hydrogels (PAAs), TGsswelled substantially after eluting the salt with ample amount ofdouble-distilled water (here and after, water specifically meansdouble-distilled water) (FIG. 4 ). We found that the linear expansionfactor of TGs was in the range of 3.0-3.5, slightly smaller than that ofthe PAAs.

Next, we tested if biomolecules, such as antibodies and FPs, can beanchored to TGs and physically pulled apart by the swelling polymernetworks. As analog to protein retention ExM (proExM), we first infusedthe cells and tissue slices with a small molecule linker (NHS-azide) sothat the residual primary amines of proteins and antibodies can becovalently bound to the polymer chains (as discussed above). We thenformed TGs in situ and digested the samples by soaking in proteinase K(proK), the same strong proteolysis used in proExM. We note that thesmall molecular linker used in our experiment can be replaced by othermolecules as long as they bind both the biomolecules of interest and theterminal or side functional groups of the TG networks.

Using this proExM analog, we embedded and expanded with TGs cultured HEKcells, which were fluorescent-labeled by antibodies (FIG. 6 ). Theimmunostained microtubules showed <4% RMS error before and after theexpansion, comparable to the previously reported value of PAA gels usinga similar metrics for the global isotropy (FIG. 6 ). These resultssuggest that TGs stay mechanical integrated during the handling and theimposed deformation is comparable to that to the PAAs.

In addition to the cultured cells, we were also able to embed and expandwith TGs thinly-sectioned mouse brain slices, which werefluorescent-labeled by endogenously expressed fluorescent proteins andsubsequent post-expansion immunostaining (FIG. 5A). We found that TGshad better preserved dye molecules that were susceptible to freeradicals, such as the cyanine dyes (FIG. 9 ). The drastically increasedfluorescence retention of red and far-red dyes demonstrates one of theadvantages to move away from the radical polymerization to betterpreserve both exogeneous and endogenous molecules (FIG. 9 ).

Finally, we used HSV-1 virions to evaluate the nanoscopic isotropy ofTGs. This is because that HSV-1 virions had (a) well-defined layeredprotein structures that had been extensively characterized byconventional high-resolution imaging methods such as electron microscopy(EM) and super-resolution microscopy, (b) the right size to validate thelocal isotropy at ˜10-100 nm length scale, and (c) establishedpurification and immobilization methods there were easily accessible tous.

Before expansion, we directly conjugated short DNA-oligos to the HSV-1envelope proteins (diameter=˜50-300 nm, width=˜50 nm) for the subsequentlabeling transfer and fluorescence read-outs. With this newdirect-labeling strategy, we were able to achieve a high-densitylabeling on the virion surface with significantly reduced size comparedwith that used in iExM, which was a combination of primary antibodiesand oligo-conjugated secondary antibodies (FIG. 11 ). The HSV-1 virionswere first iteratively expanded for two rounds (FIG. 11 ) using theTG-based (1st round: TG; 2nd and 3rd round: PAA; TG-iExM) or PAA-based(1st round: PAA; 2nd and 3rd round: PAA; PAA-iExM) iterative expansionprocess.

As result, the TG-expanded virion particles showed continuous envelopeswith significantly higher labeling density and signal-to-backgroundratio, compared with the PAA-expanded virion particles. To validate thelocal isotropy of the expansion at 10-100 nm length scale, we comparedthe widths of the virion envelope by generating an averaged singlevirion particle image from over 350 virion particles (FIG. 11 ). Wefound that the FWHM of the averaged virion envelope width was 74.5 nm(n=405) and 115.0 nm (n=357) for TG-iExM and PAA-iExM, respectively. Inaddition, we found that the mean diameter of the expanded virionsexpansion was 193.1 nm (n=429) and 210.9 nm (n=395) for TG-iExM andPAA-iExM, respectively (P<0.0001). TG-iExM showed a virion diametercloser to the previously reported value using STORM. We also note thatby applying the DAPI staining post-expansion, we were able tosimultaneously visualize the envelope proteins and DNAs of the HSV-1virions (FIG. 12 ).

Finally, we applied 3-round TG-based iterative expansion to the HSV-1virions, which yielded a 38˜40-fold expanded virion with an effectivelateral resolution of ˜7 nm (=Abbe's diffractionlimit/40=640/(2*1.15)/40=6.96 nm) and axial resolution of ˜20 nm (=axiallength of the confocal PSFs/40=˜800 nm/40=˜20 nm) (FIG. 11 ). We notethat there are a few factors that could contribute to the sparsity ofthe surface protein labeling: (a) labeling density at pre-expansionstate, (b) mesh size of the TG networks, and (c) loss of labeling duringthe iterative process. Future investigation awaits to address theseissues to achieve high labeling density for high-order expansion.

In ExM, biomolecules and fluorescent tags are anchored to a swellablehydrogel, and then pulled apart by the expanding polymer networks.Recently, multiple variants of ExM have been developed to retainproteins and RNAs in the specimen [proExM, UW version of proExM, ExFISH,ExPath, MAP, and U-ExM], and to achieve higher effective resolution byapplying the expansion process iteratively [iExM] or switching to ahighly swellable hydrogel [DMAA]. However, as discussed above, these ExMvariants suffer from nanoscopic structural inhomogeneities introduced byfree-radical polymerization which imposes an intrinsic limitation on thelocal expansion isotropy at 10-100 nm length scale. Here we show a newclass of swellable hydrogels that are tailored to overcome the localdensity fluctuations of monomers and cross-linkers and the topologicaldefects introduced by radical polymerization. By design, the polymerchain length and the cross-linking density of TGs are uniform throughoutthe gel, due to the uniform monomer size and the complementary,self-limiting polymerization mechanism between the two types of themonomers. In addition, loops and dangling ends, typical for radicalpolymerization, are substantially reduced due to the highly specific andstochiometric terminal linking of the monomers. Formed byclick-chemistry based terminal-linking of tetrahedral monomers, andtermed as tetra-gels, these non-radically polymerized hydrogels swell inwater and expand cell and tissue samples up to 3˜3.5-fold. Combining thetetra-gel-based iterative expansion and a direct-labeling strategy ofvirion envelop proteins, we have expanded HSV-1 virions 10- and 40-foldwith two or three rounds of expansions, respectively. We have found thatthe tetra-gel-expanded virions have ˜0.6 times smaller envelope widthscompared with those expanded with polyacrylamide/polyacrylate hydrogels,validating that the tetra-gels are able to capture 10-100 nm biologicalstructures with superior local isotropy. Our approach and finding serveas a guiding principle for materials design to realize ideal expansionof biological structures and open up potential applications in fieldssuch as structural biology.

REFERENCES

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While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. A hydrogel that is the product of a non-radicalpolymerization reaction between a monomer of Formula (A1):

and a monomer of Formula (B1):

wherein: each n is an integer greater than or equal to 1; each p is aninteger greater than or equal to 1; Z⁺ is a counter cation; X and Y₁ areeach crosslinkable moieties, and X and Y₁ covalently crosslink toend-link the monomers.
 2. The hydrogel of claim 1, wherein X is a moietycomprising a terminal azide group and Y₁ is a moiety comprising aterminal alkyne, and wherein X and Y₁ crosslink by copper-freeazide-alkyne cycloaddition.
 3. The hydrogel of claim 1, wherein X and Y₁crosslink by amine-NHS ester reaction.
 4. The hydrogel of claim 1,wherein X and Y₁ crosslink by maleimide-thiol reaction.
 5. The hydrogelof claim 1, wherein X and Y₁ crosslink by trans-cyclooctene(TCO)-tetrazine reaction.
 6. The hydrogel of claim 1, wherein thehydrogel is labelled.
 7. A composite comprising a biological sample andthe hydrogel of claim
 1. 8. A method of preparing the composite of claim7, comprising permeating the biological sample with a monomer of Formula(A1) or a monomer of Formula (A3), and a monomer of Formula (B1) to forma hydrogel by non-radical polymerization.
 9. A method of microscopycomprising: a. permeating the biological sample with a monomer ofFormula (A1):

and a monomer of Formula (B1):

to form a hydrogel according to claim 1 by non-radical polymerization;b. isotropically expanding the composite by contacting it with anaqueous solution; and c. viewing the expanded composite usingmicroscopy; wherein: each n is an integer greater than or equal to 1;each p is an integer greater than or equal to 1; Z⁺ is a counter cationX and Y₁ are each crosslinkable moieties, and X and Y₁ covalentlycrosslink to end-link the monomers.
 10. A method for in-situ sequencingof target nucleic acids present in a biological sample comprising thesteps of: a. attaching target nucleic acids present in the sample with amolecule linker or nucleic acid adapter; b. permeating the sample with amonomer of Formula (A1):

and a monomer of Formula (B1):

to form a hydrogel according to claim 1 by non-radical polymerizationand thereby forming a sample-hydrogel complex, wherein the smallmolecule linker or nucleic acid adaptor is attached both to the targetnucleic acids present in the sample and to the hydrogel; c. digestingproteins present in the sample; d. expanding the complex to form a firstenlarged sample; e. re-embedding the first enlarged sample in anon-swellable material to form a re-embedded complex; f. modifying thetarget nucleic acids or the nucleic acid adaptor to form a targetnucleic acids or a nucleic acid adaptor; and g. sequencing the nucleicacids present in the re-embedded complex; wherein: each n is an integergreater than or equal to 1; each p is an integer greater than or equalto 1; Z⁺ is a counter cation; X and Y₁ are each crosslinkable moieties,and X and Y₁ covalently crosslink to end-link the monomers.
 11. A methodfor enlarging a biological sample for microscopy, the method comprisingthe steps of: a) permeating a sample with a first hydrogel, wherein thesample is anchored to the swellable material; b) swelling the swellablematerial resulting in a first expanded sample; c) optionally permeatingthe first expanded sample with a second hydrogel; and d) optionallyswelling the second hydrogel resulting in a second expanded sample;wherein the first hydrogel and/or the second hydrogel is the hydrogel ofclaim 1.